Tumour necrosis factor alpha production by polymorphonuclear neutrophils treated with mouse anti-human CD19 monoclonal antibody

Polymorphonuclear neutrophils (PMNs) play pivotal roles as phagocytic cells in immune defence against bacteria and parasites, exerting their effects by production of reactive oxygen species, several cytokines, chemokines and by phagocytotic reaction. In our investigation of properties of activated PMNs, we discovered that one of the two kinds of mouse anti-human CD19 monoclonal antibodies (mAbs) clone SJ25-C1, weakly binds to freshly prepared PMNs. Moreover, the treatment of freshly prepared PMNs with anti-CD19 mAb (clone SJ25-C1) at 37 degrees C for 6 h induces the production and the secretion of tumour necrosis factor alpha (TNF alpha) by PMNs in vitro which was detectable in culture supernatants by bioassay using mouse cell line L929 cells. The concentration of TNF alpha secreted into the culture supernatant of PMNs cultured in the presence of anti-CD19 mAb (clone SJ25-C1) was higher than those of PMNs treated at 37 degrees C for 6 h with various PMN activators, such as anti-CD24 mAb, granulocytes-macrophage colony stimulation factor (GM-CSF) or interferon gamma (IFN gamma). In contrast, another clone of anti-CD19 mAb, HD37, did not bind to freshly prepared PMNs and failed to produce TNF alpha. To confirm that anti-CD19 mAb (clone SJ25-C1)-treated PMNs definitely produce TNF alpha, we measured the levels of intracellular expression of TNF alpha in PMNs permeabilized by saponin. These cells were treated with fluorescence-conjugated mouse anti-human TNF alpha mAb for detection of intracellular TNF alpha expression. Consequently, large amounts of intracellular TNF alpha were detected in PMNs treated with anti-CD19 mAb (clone SJ25-C1) but not in those treated with anti-CD19 mAb (clone HD37).     

Role of N-glycans in growth factor signaling

Secreted proteins and membrane proteins are frequently post-translationally modified by oligosaccharides. Therefore, many glycoproteins are involved in signal transduction. One example is growth factor receptors, which are membrane proteins that often contain oligosaccharides. The oligosaccharides in those growth factor receptors play crucial roles in receptor functions. An analysis of glycosyltransferase-transfectants revealed that the branching structures of oligosaccharide also serve as important determinants. For example, N-glycans of epidermal growth factor receptor (EGFR) are involved in receptor sorting, ligand binding and dimerization. The addition of a bisecting GlcNAc to N-glycans increases the endocytosis of EGFR. N-glycans of Trk, a high affinity nerve growth factor receptor, also affect its function. Thus, oligosaccharides play an important role in growth factor signaling.     

Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and the motor cortex. It has been shown that 15–20% of patients with familial ALS (FALS) have defects in the Sod1 gene, which encodes Cu,Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu,Zn-SOD, we examined the issue of whether mutated Cu,Zn-SOD affects the cell cycle. Mouse neuroblastoma Neuro-2a cells were transfected with human wild-type or mutated (G37R, G93A) Cu,Zn-SOD. Mutated, Cu,Zn-SOD-transfected cells exhibited marked retardation in cell growth and G₂/M arrest. They also displayed lower reactivity to phalloidin, indicating that the cytoskeleton was disrupted. Immunoprecipitation, two-dimensional gel electrophoresis, and Western blot analysis indicated that mutated Cu,Zn-SOD associates with actin. Similar results were obtained by in vitro incubation experiments with purified actin and mutated Cu,Zn-SOD (G93A). These results suggest that mutated Cu,Zn-SOD in FALS causes cytoskeletal changes by associating with actin, which subsequently causes G₂/M arrest and growth retardation. amyotrophic lateral sclerosis; copper; zinc superoxide dismutase; G₂/M arrest; neurodegenerative disease     

Revascularization determines volume retention and gene expression by fat grafts in mice

Autologous fat transplantation is a popular and useful technique in plastic and reconstructive surgery. The efficiency and survival of such grafts is predictable in many cases, but there are still issues to be resolved, such as how to improve graft volume retention. To address the issue of volume retention, we studied the effect of revascularization from the recipient on the size and function of adipocytes in fat grafts. Treatment of mice with TNP-470, an angiogenesis inhibitor, reduced blood flow from the recipient into the graft after subcutaneous transplantation of epididymal fat. The weight of transplanted tissues and the size of adipocytes in the grafts were significantly lower in mice treated with TNP-470 (TNP mice) than in control mice. Expression of genes for enzymes related to lipid accumulation was decreased in the grafts of TNP mice compared with control mice. Moreover, the expression of adipocyte-derived angiogenic peptides, VEGF and leptin, was significantly lower in the grafts of TNP mice than in grafts from control animals. The expression of VEGF and leptin by cultured human adipocytes was increased in the presence of conditioned medium from cultured vascular endothelial cells. These results show that the inhibition of the revascularization of fat grafts after transplantation reduces graft volume retention and cellular function. Early and adequate revascularization may be important for both the supply of nutrients and vasoactive interactions between vascular endothelial cells and adipocytes in graft.     

Membrane Potential Measurement of Protoplasts Isolated from Vigna mungo Hypocotyl Using a Fluorescent Probe, diS-C3-(5)

Membrane potentials of protoplasts isolated from Vigna mungohypocotyl segments were measured using the fluorescent probediS-C₃-(5). The fluorescence intensity changed in response tothe external K⁺ concentration. Membrane potential was estimatedto be inside negative (–85?8 mV at 0.1 mM KCl) from theNernst equation for K₊. The membrane potential was not affectedby DCCD (50 µM) or low temperature (5?C). Addition of0.5 mM Ca₂₊ to the protoplast suspension markedly depolarizedthe membrane potential, and subsequent EDTA treatment repolarizedit to the initial level. The Ca₂₊ effect on the membrane potentialmay be due to change in the permeability ratio of Cl^–to K₊. (Received December 16, 1986; Accepted April 22, 1987)     

Effects of estrus cycle on thermoregulatory responses during exercise in rats

In female rats, rectal temperature (Tre), tail vasomotor response, oxygen uptake (VO2), and carbon dioxide production (VCO2) were measured in proestrus and estrus stages during treadmill running at two different speeds at an ambient temperature (Ta) of 24 degrees C. Experiments were performed at 2.00-6.00 a.m., when the difference in Tre was greatest between the two stages; Tre at rest in the estrus stage was 0.54 degrees C higher than in the proestrus stage. In a mild warm environment, threshold Tre for a rise in tail skin temperature (Ttail) was also higher in the estrus stage than in the proestrus stage. In contrast, no difference was seen in the threshold Tre and steady state Tre at the end of exercise between proestrus and estrus stages. These values were higher at the higher work intensity. VO2 was also similar between the two stages, except in the second 5 min after the beginning of exercise, when VO2 was greater and Tre rose more steeply in the proestrus stage. These data indicate that deep body temperature during exercise is regulated at a certain level depending on the work intensity and is not influenced by the estrus cycle.     

Toad egg-jelly as a source of divalent cations essential for fertilization

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boer's solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.     

Bovine milk-derived α-lactalbumin prevents hepatic fibrosis induced by dimethylnitrosamine via nitric oxide pathway in rats

The present study was designed to evaluate the hepatoprotective potential of α-lactalbumin (αLA) against dimethylnitrosamine (DMN)-induced toxic insults in the rat liver. The liver damage was induced in rats by the repeated administration of DMN (10 mg/kg, i.p.) on three consecutive days per week for three weeks. The rats were maintained on either a standard AIN-93 M or αLA-enriched diet starting one week before the DMN injection until the termination of the experiment. The DMN treatment produced a progressive increase in the plasma markers (aspartate aminotransferase, alanine aminotransferase, total bililbin, hyarulonic acid, and matrix metalloproteinase-2) in 28 days after the first DMN injection. Dietary treatment with αLA significantly reduced the DMN-induced damage toward normalcy. N^Ｇ-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, significantly attenuated the hepatoprotective effect of αLA. These findings show that αLA has a marked suppressive effect on hepetic fibrosis through a nitric oxide-mediated mechanism.